Medical recombinant collagen repair gel and preparation method thereof

Abstract

The invention relates to medical recombinant collagen repair gel and a preparation method thereof, and belongs to the technical field of recombinant collagen. Through the synergistic effect of the collagen, the chitosan, the polylactic acid, the nanocellulose, the vascular endothelial growth factor, the anti-inflammatory drug and the cell adhesion peptide, the tissue repair effect is remarkably improved. Through a gas foaming method, staged freezing and freeze drying, multistage pore size distribution is generated, gel with a porous structure is formed, and cell permeation and tissue regeneration are facilitated; in addition, the permeability and the drug loading capacity of the gel are improved, the cell adhesion peptides, the growth factors and the anti-inflammatory drugs are better adsorbed to the gel, functional slow release is achieved, and the repairing effect is further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of recombinant collagen, and relates to a medical recombinant collagen repair gel and a preparation method thereof. Background Art

[0002] Collagen, as the main component of the extracellular matrix of the human body, plays an irreplaceable role in tissue repair and regeneration. In recent years, medical recombinant collagen repair gels have gradually become a research hotspot in the fields of wound repair, burn treatment, and medical aesthetics due to their excellent biocompatibility, controllable degradability, and low immunogenicity. Compared with traditional animal-derived collagen, recombinant collagen expresses specific functional domains through genetic engineering techniques, can precisely simulate the amino acid sequence of natural collagen, significantly reduce the risk of immune rejection, and avoid the problem of virus contamination at the same time. Such gels can simulate the extracellular microenvironment through a three-dimensional network structure loaded with recombinant collagen, promote cell adhesion, proliferation, and migration, and accelerate wound healing. However, its clinical application still faces technical bottlenecks such as insufficient gel mechanical properties, poor stability, and low sustained-release efficiency of active ingredients, and it is urgently needed to be broken through by optimizing the preparation process and functional design.

[0003] In the prior art, the preparation of collagen gels is mostly based on collagen extracted from animal sources. However, in the past, most collagen scaffolds were extracted from the tendons of cows and horses, and then cross-linked with cross-linking agents such as glutaraldehyde to reconstruct the collagen scaffold. However, due to problems such as the risk of immunogenicity, instability, and inconsistent quality between batches of collagen derived from natural tissues, in recent years, recombinant collagen technology has developed rapidly. Recombinant collagen is synthesized in microorganisms or other expression systems by genetic engineering means, which can avoid the immunogenicity problem of natural collagen and improve the controllability and purity of production at the same time. Therefore, medical repair gels developed based on recombinant collagen not only have the advantages of natural collagen, but also have more excellent physical and chemical properties and biological safety, and have become a research hotspot in the field of tissue repair.

[0004] Extracting collagen from animal sources is not only expensive but also destroys the natural structure between collagen bundles, making it difficult to meet the mechanical and biological performance requirements of bone substitute materials. At the same time, it may cause high resistance to tissues. With the development of genetic engineering technology, the preparation of recombinant collagen has gradually become the mainstream. For example, recombinant human type III collagen is produced using the Pichia pastoris expression system and formed into a gel by ionic crosslinking method. However, its gel network is loose and it is difficult to maintain long-term structural stability. In addition, in the prior art, a blending modification strategy is often adopted to compound recombinant collagen with hyaluronic acid and enhance the water retention of the gel by hydrogen bond interaction, but the problem of excessive local concentration caused by the burst release of active ingredients has not been solved. Some studies have tried to introduce photo-crosslinking technology to achieve photo-curing molding of the gel through methacrylation modification. However, the photoinitiator may cause cytotoxic reactions, and the complex modification process is likely to damage the natural conformation of collagen. Therefore, developing a preparation method for a highly efficient, safe and controllable medical recombinant collagen repair gel has become a key problem to be solved urgently. Summary of the Invention

[0005] The present invention relates to a medical recombinant collagen repair gel and a preparation method thereof, belonging to the technical field of recombinant collagen. Through the synergistic effect of collagen, chitosan, polylactic acid, nanocellulose, vascular endothelial growth factor, anti-inflammatory drugs and cell adhesion peptides, the present invention significantly improves the tissue repair effect. Through gas foaming method, staged freezing and freeze-drying, a multi-level pore size distribution is generated to form a gel with a porous structure, which is beneficial to cell penetration and tissue regeneration; and the permeability and drug loading capacity of the gel are improved, and the cell adhesion peptides, growth factors and anti-inflammatory drugs are better adsorbed on the gel to achieve functional slow release, further improving the repair effect.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] A medical recombinant collagen repair gel, comprising the following raw materials in parts by weight: 40-50 parts of collagen, 15-20 parts of chitosan, 10-15 parts of polylactic acid, 5-10 parts of nanocellulose, 1-2 parts of vascular endothelial growth factor, 1-3 parts of anti-inflammatory drugs, 1-2 parts of cell adhesion peptides.

[0008] As a preferred technical solution of the present invention, the anti-inflammatory drug is any one of dexamethasone, indomethacin, ibuprofen; the cell adhesion peptide is any one of RGD peptide, fibronectin.

[0009] Furthermore, the preparation method of the medical recombinant collagen repair gel comprises the following steps:

[0010] (1) Dissolve collagen in a 0.1M ice acetic acid solution, stir until completely dissolved to obtain a collagen solution. Then dissolve chitosan in a 0.1M ice acetic acid solution, stir until transparent to obtain a chitosan solution. Drop the collagen solution into the chitosan solution, stir evenly to form a mixed solution. Disperse polylactic acid in absolute ethanol and ultrasonically treat it for 10 - 30 min to obtain a polylactic acid solution. Drop the polylactic acid solution into the mixed solution and stir evenly to prepare a matrix solution;

[0011] (2) Disperse nanocellulose in deionized water, ultrasonically treat it for 10 - 20 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 6.5 - 7.4 with 1M sodium hydroxide solution, and add a crosslinking agent and stir evenly to prepare a basic gel solution;

[0012] (3) Place the basic gel solution in a sealed container, inject foaming gas through a micro gas injection device, add a crosslinking agent until the concentration is 10 - 20 mmol / L, and stir at 4°C for 12 hours, then perform vacuum treatment at -10 to -100 kPa for 30 min to remove residual gas;

[0013] (4) Freeze the gas - foamed gel solution at -80°C for 1 - 2 hours, then freeze it in an environment of -20°C for 4 - 6 hours, and obtain the basic gel by vacuum freeze - drying;

[0014] (5) Add a crosslinking agent to a cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 20 - 40 min, add the basic gel, soak for 3 - 5 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptide and crosslinking agent to obtain a cell adhesion gel;

[0015] (6) Soak the cell adhesion gel in a solution containing 10 - 50 ng / mL vascular endothelial growth factor and 10 - 100 μg / mL anti - inflammatory drug, allow the growth factor and anti - inflammatory drug to enter the pores of the gel by diffusion adsorption, wash with PBS at pH 7.4 to remove unbound growth factor, and vacuum - dry at 10 - 40°C for 2 - 4 h;

[0016] (7) Add 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide to a concentration of 5 - 15 mmol / L and N - hydroxysuccinimide to a concentration of 2 - 8 mmol / L, stir at 1 - 9°C for 10 - 14 h for crosslinking to obtain a medical recombinant collagen repair gel.

[0017] As a preferred technical solution of the present invention, in step (1), the concentration of the collagen solution is 1 - 2 wt%; the concentration of the chitosan solution is 1 - 2 wt%; the concentration of the polylactic acid solution is 5 - 10 wt%.

[0018] As a preferred technical solution of the present invention, the cross-linking agent in step (2) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of the nanocellulose, deionized water, matrix solution, and cross-linking agent is 1-5:70-85:10-20:0.5-1.

[0019] As a preferred technical solution of the present invention, the cross-linking agent in step (3) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the foaming gas is carbon dioxide or nitrogen; the pressure for injecting the foaming gas is 50-200 kPa, and the injection time is 5-15 min.

[0020] As a preferred technical solution of the present invention, the vacuum freeze-drying conditions in step (4) are -50°C, the vacuum degree is 5-15 Pa, and the vacuum freeze-drying time is 24-48 h.

[0021] As a preferred technical solution of the present invention, the cross-linking agent in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide or N-hydroxysuccinimide; the mass ratio of the cell adhesion peptide solution, cross-linking agent, basic gel, and PBS is 1:0.01-0.2:5-15:15-25.

[0022] As a preferred technical solution of the present invention, the mass ratio of the cell adhesion gel vascular endothelial growth factor and anti-inflammatory drug solution, and PBS in step (6) is 1:3-5:8-12.

[0023] The beneficial effects of the present invention:

[0024] (1) The present invention combines various biomaterials and active molecules such as collagen, chitosan, polylactic acid, nanocellulose, vascular endothelial growth factor, anti-inflammatory drugs, and cell adhesion peptides, endowing the gel with multifunctionality. Collagen provides good biocompatibility and cell adhesion ability, chitosan has antibacterial properties and promotes wound healing, while polylactic acid provides mechanical strength and controllable degradation performance. The introduction of nanocellulose further enhances the mechanical strength and structural stability of the gel, while forming a three-dimensional support framework, providing an ideal microenvironment for cell adhesion and proliferation. This multi-component composite design not only significantly improves the biological activity and functionality of the gel, but also can adapt to different tissue repair needs by regulating the proportion of each component.

[0025] (2) By using carbon dioxide or nitrogen for foaming treatment, the present invention makes the pore distribution more uniform, significantly improves the permeability and drug loading capacity of the gel, and thus realizes the controlled release of growth factors and anti-inflammatory drugs. At the same time, by freezing in stages, the gel forms a three-dimensional porous structure with gradient pore sizes, enhancing the cell permeability and tissue regeneration ability of the gel. In addition, the cross-linking technology uses 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, which can effectively cross-link collagen and chitosan, ensuring the stable mechanical properties of the gel while maintaining its biocompatibility. Detailed implementation manners

[0026] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following, in combination with embodiments, details the specific implementation manners, structures, features and their effects according to the present invention as follows.

[0027] Example 1

[0028] A medical recombinant collagen repair gel comprises the following raw materials in parts by weight: 45 parts of collagen, 18 parts of chitosan, 13 parts of polylactic acid, 7 parts of nanocellulose, 1.5 parts of vascular endothelial growth factor, 2 parts of anti-inflammatory drug, and 1.5 parts of cell adhesion peptide.

[0029] The anti-inflammatory drug is dexamethasone; the cell adhesion peptide is RGD peptide.

[0030] The preparation method of the medical recombinant collagen repair gel comprises the following steps:

[0031] (1) Dissolve collagen in a 0.1M glacial acetic acid solution, stir until completely dissolved to obtain a collagen solution, then dissolve chitosan in a 0.1M glacial acetic acid solution, stir until transparent to obtain a chitosan solution, drop the collagen solution into the chitosan solution, stir evenly to form a mixed solution, disperse polylactic acid in absolute ethanol and ultrasonically treat for 20 min to obtain a polylactic acid solution, and drop the polylactic acid solution into the mixed solution, stir evenly to prepare a matrix solution;

[0032] (2) Disperse nanocellulose in deionized water, ultrasonically treat for 15 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 7.0 with a 1M sodium hydroxide solution, add a cross-linking agent and stir evenly to prepare a basic gel solution;

[0033] (3) Place the basic gel solution in a sealed container, inject a foaming gas through a micro gas injection device, add a cross-linking agent until the concentration is 15 mmol / L, and stir at 4°C for 12 hours, then perform a -50 kPa vacuum treatment for 30 min to remove the residual gas;

[0034] (4) Freeze the gas-foamed gel solution at -80°C for 1.5 hours, then freeze it in an environment of -20°C for 5 hours, and obtain the basic gel through vacuum freeze-drying.

[0035] (5) Add a crosslinking agent to the cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 30 min, add the basic gel, soak for 4 h, and then wash with PBS at pH 7.4 to remove the unbound cell adhesion peptide and crosslinking agent to obtain the cell adhesion gel.

[0036] (6) Immerse the cell adhesion gel in a solution containing 30 ng / mL vascular endothelial growth factor and 55 μg / mL anti-inflammatory drug, and allow the growth factor and anti-inflammatory drug to enter the pores of the gel through diffusion adsorption. Wash with PBS at pH 7.4 to remove the unbound growth factor, and vacuum-dry at 20°C for 3 h.

[0037] (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 10 mmol / L and N-hydroxysuccinimide to a concentration of 5 mmol / L, stir at 4°C for 12 h for crosslinking to obtain the medical recombinant collagen repair gel.

[0038] In step (1), the concentration of the collagen solution is 1.5 wt%; the concentration of the chitosan solution is 1.5 wt%; the concentration of the polylactic acid solution is 8 wt%.

[0039] In step (2), the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the nanocellulose, deionized water, matrix solution, and crosslinking agent is 3:80:15:0.8.

[0040] In step (3), the crosslinking agent is N-hydroxysuccinimide; the foaming gas is carbon dioxide; the injection pressure of the foaming gas is 150 kPa, and the injection time is 10 min.

[0041] In step (4), the vacuum freeze-drying conditions are -50°C, the vacuum degree is 10 Pa, and the vacuum freeze-drying time is 36 h.

[0042] In step (5), the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the cell adhesion peptide solution, crosslinking agent, basic gel, and PBS is 1:0.1:10:20.

[0043] In step (6), the mass ratio of the cell adhesion gel, vascular endothelial growth factor and anti-inflammatory drug solution, and PBS is 1:4:10.

[0044] Example 2

[0045] A medical recombinant collagen repair gel, comprising the following raw materials in parts by weight: 40 parts of collagen, 15 parts of chitosan, 10 parts of polylactic acid, 5 parts of nanocellulose, 1 part of vascular endothelial growth factor, 1 part of anti-inflammatory drug, and 1 part of cell adhesion peptide.

[0046] The anti-inflammatory drug is indomethacin; the cell adhesion peptide is fibronectin.

[0047] The preparation method of the medical recombinant collagen repair gel comprises the following steps:

[0048] (1) Dissolve collagen in a 0.1M glacial acetic acid solution, stir until completely dissolved to obtain a collagen solution, then dissolve chitosan in a 0.1M glacial acetic acid solution, stir until transparent to obtain a chitosan solution, drop the collagen solution into the chitosan solution, stir evenly to form a mixed solution, disperse polylactic acid in absolute ethanol and ultrasonically treat for 10 min to obtain a polylactic acid solution, drop the polylactic acid solution into the mixed solution, and stir evenly to prepare a matrix solution;

[0049] (2) Disperse nanocellulose in deionized water, ultrasonically treat for 10 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 6.5 with a 1M sodium hydroxide solution, and add a crosslinking agent and stir evenly to prepare a basic gel solution;

[0050] (3) Place the basic gel solution in a closed container, inject foaming gas through a micro gas injection device, add a crosslinking agent until the concentration is 10 mmol / L, and stir at 4°C for 12 hours, and perform vacuum treatment at -10 kPa for 30 min to remove residual gas;

[0051] (4) Freeze the gas-foamed gel solution at -80°C for 1 hour, then freeze it in a -20°C environment for 4 hours, and prepare a basic gel by vacuum freeze-drying;

[0052] (5) Add a crosslinking agent to a cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 20 min, add the basic gel, soak for 3 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptide and crosslinking agent to prepare a cell adhesion gel;

[0053] (6) Soak the cell adhesion gel in a solution containing 10 ng / mL vascular endothelial growth factor and 10 μg / mL anti-inflammatory drug, and allow the growth factor and anti-inflammatory drug to enter the pores of the gel by diffusion adsorption, wash with PBS at pH 7.4 to remove unbound growth factor, and perform vacuum drying at 10°C for 2 h;

[0054] (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 5 mmol / L and N-hydroxysuccinimide to a concentration of 2 mmol / L, and stir at 1 °C for 10 h for crosslinking to obtain a medical recombinant collagen repair gel.

[0055] In step (1), the concentration of the collagen solution is 1-2 wt%; the concentration of the chitosan solution is 1 wt%; the concentration of the polylactic acid solution is 5 wt%.

[0056] In step (2), the crosslinking agent is N-hydroxysuccinimide; the mass ratio of the nanocellulose, deionized water, matrix solution, and crosslinking agent is 1:70:10:0.5.

[0057] In step (3), the crosslinking agent is N-hydroxysuccinimide; the foaming gas is nitrogen; the injection pressure of the foaming gas is 50 kPa, and the injection time is 15 min.

[0058] In step (4), the vacuum freeze-drying conditions are -50 °C, the vacuum degree is 15 Pa, and the vacuum freeze-drying time is 24 h.

[0059] In step (5), the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the cell adhesion peptide solution, crosslinking agent, basic gel, and PBS is 1:0.01:5:15.

[0060] In step (6), the mass ratio of the cell adhesion gel vascular endothelial growth factor and anti-inflammatory drug solution, and PBS is 1:3:8.

[0061] Example 3

[0062] A medical recombinant collagen repair gel, comprising the following raw materials in parts by weight: 50 parts of collagen, 20 parts of chitosan, 15 parts of polylactic acid, 10 parts of nanocellulose, 2 parts of vascular endothelial growth factor, 3 parts of anti-inflammatory drug, and 2 parts of cell adhesion peptide.

[0063] The anti-inflammatory drug is ibuprofen; the cell adhesion peptide is RGD peptide.

[0064] The preparation method of the medical recombinant collagen repair gel comprises the following steps:

[0065] (1) Dissolve collagen in a 0.1 M glacial acetic acid solution, stir until completely dissolved to obtain a collagen solution, then dissolve chitosan in a 0.1 M glacial acetic acid solution, stir until transparent to obtain a chitosan solution, drop the collagen solution into the chitosan solution, stir evenly to form a mixed solution, disperse polylactic acid in absolute ethanol and ultrasonically treat for 30 min to obtain a polylactic acid solution, and drop the polylactic acid solution into the mixed solution, stir evenly to prepare a matrix solution;

[0066] (2) Disperse nanocellulose in deionized water, ultrasonically treat for 20 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 7.4 with 1 M sodium hydroxide solution, and add a crosslinking agent and stir evenly to obtain a basic gel solution;

[0067] (3) Place the basic gel solution in a sealed container, inject foaming gas through a micro gas injection device, add a crosslinking agent until the concentration is 20 mmol / L, and stir at 4 °C for 12 hours, then perform vacuum treatment at -100 kPa for 30 min to remove residual gas;

[0068] (4) Freeze the gas-foamed gel solution at -80 °C for 2 hours, then freeze it at -20 °C for 6 hours, and obtain the basic gel by vacuum freeze-drying;

[0069] (5) Add a crosslinking agent to the cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 40 min, add the basic gel, soak for 5 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptide and crosslinking agent to obtain a cell adhesion gel;

[0070] (6) Immerse the cell adhesion gel in a solution containing 50 ng / mL vascular endothelial growth factor and 100 μg / mL anti-inflammatory drug, and allow the growth factor and anti-inflammatory drug to enter the pores of the gel by diffusion adsorption, wash with PBS at pH 7.4 to remove unbound growth factor, and vacuum dry at 40 °C for 4 h;

[0071] (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 15 mmol / L and N-hydroxysuccinimide to a concentration of 8 mmol / L, and stir at 9 °C for 14 h for crosslinking to obtain a medical recombinant collagen repair gel.

[0072] In step (1), the concentration of the collagen solution is 2 wt%; the concentration of the chitosan solution is 2 wt%; the concentration of the polylactic acid solution is 10 wt%.

[0073] In step (2), the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the nanocellulose, deionized water, matrix solution, and crosslinking agent is 5:85:20:1.

[0074] In step (3), the crosslinking agent is N-hydroxysuccinimide; the foaming gas is carbon dioxide; the pressure for injecting the foaming gas is 200 kPa, and the injection time is 15 min.

[0075] In step (4), the vacuum freeze-drying conditions are -50 °C, the vacuum degree is 15 Pa, and the vacuum freeze-drying time is 48 h.

[0076] The crosslinking agent described in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of the cell adhesion peptide solution, crosslinking agent, basic gel, and PBS is 1:0.2:15:25.

[0077] The mass ratio of the cell adhesion gel vascular endothelial growth factor, anti-inflammatory drug solution, and PBS described in step (6) is 1:5:12.

[0078] Comparative Example 1

[0079] Based on Example 1, chitosan is not added to the raw materials, and the addition amount of polylactic acid is changed to 31 parts, and the rest is the same as Example 1.

[0080] Comparative Example 2

[0081] Based on Example 1, polylactic acid is not added to the raw materials, and the addition amount of chitosan is changed to 31 parts, and the rest is the same as Example 1.

[0082] Comparative Example 3

[0083] Based on Example 1, foaming gas is not injected, and the rest is the same as Example 1.

[0084] Comparative Example 4

[0085] Based on Example 1, step (4) is changed to freeze the gas-foamed gel solution at -80°C for 6.5 hours, and the basic gel is prepared by vacuum freeze-drying, and the rest is the same as Example 1.

[0086] Comparative Example 5

[0087] Based on Example 1, step (4) is changed to freeze the gas-foamed gel solution at -20°C for 6.5 hours, and the basic gel is prepared by vacuum freeze-drying, and the rest is the same as Example 1.

[0088] Performance test:

[0089] Repair test: The medical recombinant collagen repair gels prepared in Application Examples 1-3 and Comparative Application Examples 1-5 were respectively diluted into 5% concentration solutions with a medium containing 2% serum. The blank control group was a medium containing 2% serum, and the positive control group was a medium containing 10% serum.

[0090] Inoculate BJ cells in the logarithmic growth phase into a 24-well plate, and the inoculation density is 2.5×10 5cells / well. Incubate in a 37°C carbon dioxide incubator for 24 h. After the cells are almost confluent, use a pipette tip to make a cross in the 24-well plate, then wash once with PBS. Add the diluted samples to the wells, 500 μL per well, and incubate in a 37°C carbon dioxide incubator until the observation time set for the experiment. At the set time, observe and measure the width of the scratch with a microscope.

[0091] The calculation formula for the scratch healing rate is as follows:

[0092]

[0093] Test results:

[0094]

[0095] It can be seen from the test results that through the synergistic effect of collagen, chitosan, polylactic acid, nanocellulose, vascular endothelial growth factor, anti-inflammatory drugs and cell adhesion peptides, the present invention improves the tissue repair effect. And through gas foaming method, staged freezing and freeze-drying, it is beneficial to cell penetration and tissue regeneration, further improving the repair effect.

[0096] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, any indirect modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A medical recombinant collagen repair gel, characterized in that: It comprises the following raw materials in parts by weight: 40-50 parts of collagen, 15-20 parts of chitosan, 10-15 parts of polylactic acid, 5-10 parts of nanocellulose, 1-2 parts of vascular endothelial growth factor, 1-3 parts of anti-inflammatory drug, and 1-2 parts of cell adhesion peptide.

2. The recombinant collagen repair gel according to claim 1, characterized in that: The anti-inflammatory drug is any one of dexamethasone, indomethacin, and ibuprofen; the cell adhesion peptide is any one of RGD peptide and fibronectin.

3. A preparation method of the medical recombinant collagen repair gel as described in claim 1, characterized in that: It comprises the following steps: (1) Dissolve collagen in 0.1M glacial acetic acid solution, stir until completely dissolved to obtain a collagen solution. Then dissolve chitosan in 0.1M glacial acetic acid solution, stir until transparent to obtain a chitosan solution. Drop the collagen solution into the chitosan solution, stir evenly to form a mixed solution. Disperse polylactic acid in absolute ethanol and ultrasonically treat it for 10-30 min to obtain a polylactic acid solution. Drop the polylactic acid solution into the mixed solution and stir evenly to prepare a matrix solution. (2) Disperse nanocellulose in deionized water, ultrasonically treat it for 10-20 min to mix evenly to form a suspension, drop it into the matrix solution, stir evenly, adjust the pH to 6.5-7.4 with 1M sodium hydroxide solution, and add a cross-linking agent and stir evenly to prepare a basic gel solution. (3) Place the basic gel solution in a sealed container, inject foaming gas through a micro gas injection device, add a cross-linking agent until the concentration is 10-20 mmol / L, and stir for 12 hours at 4°C, then perform vacuum treatment at -10 to -100 kPa for 30 min to remove residual gas. (4) Freeze the gas-foamed gel solution at -80°C for 1-2 hours, then freeze it in an environment of -20°C for 4-6 hours, and obtain a basic gel by vacuum freeze-drying. (5) Add a cross-linking agent to a cell adhesion peptide solution with a concentration of 1 mg / mL, stir for 20-40 min, add the basic gel, soak for 3-5 h, and then wash with PBS at pH 7.4 to remove unbound cell adhesion peptide and cross-linking agent to prepare a cell adhesion gel. (6) Soak the cell adhesion gel in a solution containing 10-50 ng / mL vascular endothelial growth factor and 10-100 μg / mL anti-inflammatory drug, and allow the growth factor and anti-inflammatory drug to enter the pores of the gel by diffusion adsorption. Wash with PBS at pH 7.4 to remove unbound growth factor, and vacuum dry at 10-40°C for 2-4 h. (7) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a concentration of 5-15 mmol / L and N-hydroxysuccinimide to a concentration of 2-8 mmol / L, stir at 1-9°C for 10-14 h for cross-linking to obtain a medical recombinant collagen repair gel.

4. The preparation method of a medical recombinant collagen repair gel according to claim 3, wherein: In step (1), the concentration of the collagen solution is 1-2 wt%; the concentration of the chitosan solution is 1-2 wt%; the concentration of the polylactic acid solution is 5-10 wt%.

5. The preparation method of a medical recombinant collagen repair gel according to claim 3, wherein: The cross-linking agent described in step (2) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of the nanocellulose, deionized water, matrix solution, and cross-linking agent is 1-5:70-85:10-20:0.5-1.

6. The preparation method of a medical recombinant collagen repair gel according to claim 3, characterized in that: The cross-linking agent described in step (3) is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the foaming gas is carbon dioxide or nitrogen; the injection pressure of the foaming gas is 50-200 kPa, and the injection time is 5-15 min.

7. The preparation method of a medical recombinant collagen repair gel according to claim 3, characterized in that: The vacuum freeze-drying conditions in step (4) are -50°C, the vacuum degree is 5-15 Pa, and the vacuum freeze-drying time is 24-48 h.

8. The preparation method of a medical recombinant collagen repair gel according to claim 3, characterized in that: The cross-linking agent described in step (5) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide or N-hydroxysuccinimide; the mass ratio of the cell adhesion peptide solution, cross-linking agent, basic gel, and PBS is 1:0.01-0.2:5-15:15-25.

9. The preparation method of a medical recombinant collagen repair gel according to claim 3, wherein: The mass ratio of the cell adhesion gel vascular endothelial growth factor and anti-inflammatory drug solution, and PBS in step (6) is 1:3-5:8-12.

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